Hybrid thymus for inducing xenograft tolerance and restoring immunocompetence and thymic function, methods for making and using same

Abstract

The present disclosure relates to the generation of pig-human hybrid thymic tissue and its use to induce tolerance in xenotransplantation, which can also be used to restore or induce immunocompetence in a recipient and restore or promote thymus dependency for the development of T cell progenitors into mature functional T cells in the recipient. [Selected Figure] Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO OTHER APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 734,019, filed September 20, 2018, which is incorporated herein by reference in its entirety.

[0002] Government Interest Statement This invention was made with government support under grants AI084903, AI045897, and AI106697 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present disclosure relates to generating a pig-human hybrid thymus and using the hybrid thymus to induce tolerance in xenotransplantation. [Background technology]

[0004] Background of the Invention A severe shortage of allogeneic donors limits the number of organ transplants currently performed. This supply-demand discrepancy could be corrected by the use of organs from other species (xenografts). Given the ethical issues and impracticalities associated with the use of nonhuman primates, pigs are considered the most suitable donor species for humans. Their organ size and physiological similarities to humans, along with the ability to rapidly raise and inbred pigs, make them particularly suitable for genetic modification that could improve their ability to serve as transplant donors for humans (Sachs, Path. Biol. 42:217-219, 1994; Piedrahita et al., Am. J. Transplant, 4 Suppl. 6:43-50, 2004).

[0005] Despite recent advances, the immune response to xenografts remains strong, limiting their clinical use. Although transplantation combined with nonspecific immunosuppressive therapy is associated with high initial graft tolerance, the main limitation to the success of clinical organ transplantation has been late graft loss, largely due to chronic rejection of the transplant. Furthermore, immunosuppressive therapy often results in adverse side effects or increases the risk of infection. Thus, methods for controlling the immune response to xenografts would significantly improve their applicability.

[0006] Genetically engineered pigs lacking the Gal gene avoid common rejection in non-human primates due to natural anti-Galα1-3Gal (Gal) antibodies. Cooper D. A brief history of cross-species organ transplantation. Proc (Baylor Univ Med Cent). 2012 Jan;25(1):49-57. Despite this progress, T cell-dependent antibodies can recognize other pig specificities that cause rejection. Yang YG, Sykes M. Xenotransplantation: current status and a perspective on the future. Nat Rev Immunol. 2007 Jul;7(7):519-31. Extended T cell suppression. Pig xenografts survive in non-human primates, but such treatment is highly toxic. Yamada K, Sykes M, Sachs DH. Tolerance in xenotransplantation. Curr Opin Organ Transplant. 2017 Dec;22(6):522-528.

[0007] Another approach to T cell suppression is tolerance induction. Xenograft tolerance approaches include mixed chimerism induction and pig thymus transplantation.

[0008] Mixed chimerism can induce tolerance to the donor at the level of T cells, B cells, and natural killer (NK) cells in the recipient. Griesemer A., ​​Yamada K., and Sykes M., Xenotransplantation: Immunological hurdles and progress toward tolerance, Immunol Rev. 2014;258(1):241-258. Sachs DH, Kawai T., and Sykes M., Cold Spring Harb. Perspect. Med. 2014;4:a015529.

[0009] Thymic xenotransplantation can also induce robust tolerance. Kalscheuer H, Onoe T, Dahmani A, Li HW, Holzl M, Yamada K, Sykes M. Xenograft Tolerance and Immune Function of Human T Cells Developing in Pig Thymus Xenografts. J Immunol. 2014 Apr 1;192(7):3442-50. Pig thymus grafts can generate diverse and functional human T cell repertoires in mice that are specifically unresponsive to the donor pig in vitro. Shimizu I, Fudaba Y, Shimizu A, Yang Y, and Sykes M. Comparison of human T cell repertoire generated in xenogeneic porcine and human thymus grafts. Transplantation. 2008 Aug 27;86(4):601-610. Nikolic B, J.P. Gardner, D.T. Scadden, J.S. Arn, D.H. Schachs, and M. Sykes. Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. J.Immunol. 1999 Mar 15;162:3402-3407.Habiro K, Sykes M, Yang YG. Induction of human T-cell tolerance to pig xenoantigens via thymus transplantation in mice with an established human immune system. Am J Transplant. 2009 Jun;9(6):1324-9. Extension of this technique from a pig to a baboon model achieved sustained pig kidney xenograft survival in non-human primates. Yamada K, Yazawa K, Shimizu A, Iwanaga T, Hisashi Y, Nuhn M, O'Malley P, Nobori S, Vagefi PA, Patience C, Fishman J, Cooper DK, Hawley RJ, Greenstein J, Schuurman HJ, Awwad M, Sykes M, Sachs DH. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med. 2005 Jan;11(1):32-4.

[0010] However, remaining limitations include suboptimal recipient immune function that cannot efficiently recognize foreign antigens; suboptimal survival, homeostasis, and inefficient elimination of autoreactive T cells; and a lack of positive selection of regulatory T cells to prevent autoimmunity. Summary of the Invention

[0011] The present disclosure provides a method for inducing tolerance in a recipient mammal of a first species to a graft obtained from a donor mammal of a second species, the method comprising: (a) introducing hybrid thymic tissue from a second species, the hybrid thymic tissue comprising thymic epithelial cells from a first species into a recipient mammal; and (b) transplanting the graft from the donor mammal into the recipient mammal; The present invention provides a method comprising: [Means for solving the problem]

[0012] In some embodiments, thymic function is essentially absent in the recipient mammal prior to performing step (a). In some embodiments, the recipient mammal is a primate, and in some embodiments, it is a human. In some embodiments, the donor mammal is a pig, and in some embodiments, it is a minipig.

[0013] In some embodiments, the thymus tissue from the donor mammal is fetal thymus tissue. In some embodiments, the thymus tissue from the donor mammal is neonatal thymus tissue.

[0014] In some embodiments, the thymic epithelial cells from the recipient mammal are obtained from the thymus of the recipient mammal. In some embodiments, the thymic epithelial cells from the recipient mammal are generated from induced pluripotent stem cells (iPSCs) of the recipient mammal. In some embodiments, the thymic epithelial cells from the recipient mammal species are generated from embryonic stem cells that share HLA alleles with the recipient mammal. In some embodiments, the embryonic stem cells are genetically engineered to share HLA alleles with the recipient mammal.

[0015] In some embodiments, the hybrid thymus tissue is transplanted into a recipient mammal in step (a). In some embodiments, step (a) is performed before or simultaneously with step (b).

[0016] In some embodiments, hybrid thymic tissue is generated by introducing thymic epithelial cells from a recipient mammal into thymic tissue from a donor mammal. In some embodiments, hybrid thymic tissue is generated by injecting thymic epithelial cells from a first species of recipient mammal into thymic tissue from a second species of donor mammal. In some embodiments, the method further comprises administering hematopoietic stem cells (HSCs) to the recipient mammal. In some embodiments, the graft comprises cells, tissues, or organs.

[0017] In a further embodiment, the hybrid thymus tissue is prepared by the following steps: (i) treating thymus tissue from a second species of donor mammal with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from a first type of recipient mammal into the 2DG-treated thymic tissue; The method is produced by a method comprising:

[0018] In some embodiments, thymic epithelial cells from a first species are suspended in Matrigel before being injected into the 2DG-treated thymic tissue.

[0019] The present disclosure also provides a method of restoring or inducing immunocompetence in a recipient mammal of a first species, the method comprising introducing into the recipient mammal of the first species thymic tissue, the hybrid thymic tissue comprising thymic epithelial cells from a donor mammal of a second species.

[0020] The present disclosure also provides a method for restoring or promoting thymic dependency of T cell progenitor cells that develop into mature functional T cells in a recipient mammal of a first species, comprising introducing thymic tissue from a donor mammal of a second species, hybrid thymic tissue comprising thymic epithelial cells from the first species, into the recipient mammal of the first species.

[0021] In some embodiments of these methods, thymic function is essentially absent in the recipient mammal prior to the transferring step. In some embodiments, the recipient mammal is thymectomized prior to the transferring step. In some embodiments, the recipient mammal has an immune disorder.

[0022] In some embodiments, the donor mammal is a pig, and in some embodiments, the pig is a minipig. In some embodiments, the recipient mammal is a primate. In some embodiments, the recipient mammal is a human.

[0023] In some embodiments, the thymus tissue from the donor mammal is fetal thymus tissue. In some embodiments, the thymus tissue from the donor mammal is neonatal thymus tissue.

[0024] In some embodiments, the thymic epithelial cells are obtained from the thymus of the recipient mammal. In some embodiments, the thymic epithelial cells are generated from induced pluripotent stem cells (iPSCs) of the recipient mammal. In some embodiments, the thymic epithelial cells are generated from embryonic stem cells that share HLA alleles with the recipient mammal. In some embodiments, the embryonic stem cells are genetically engineered to share HLA alleles with the recipient mammal.

[0025] In some embodiments, the hybrid thymus tissue is transplanted into a recipient mammal.

[0026] In some embodiments, hybrid thymic tissue is generated by introducing thymic epithelial cells from a first species into thymic tissue from a donor mammal of a second species. In some embodiments, hybrid thymic tissue is generated by injecting thymic epithelial cells from a first species into thymic tissue from a donor mammal of a second species.

[0027] In some embodiments, the hybrid thymus tissue is prepared by the following steps: (i) treating thymus tissue from a second species of donor mammal with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from the first species into the 2DG-treated thymic tissue; The method is produced by a method comprising:

[0028] In some embodiments, thymic epithelial cells from a first species are suspended in Matrigel before being injected into the 2DG-treated thymic tissue.

[0029] The present disclosure also provides isolated hybrid thymic tissue comprising thymic epithelial cells from a first mammalian species and thymic tissue from a second mammalian species, as well as methods of making the hybrid thymic tissue.

[0030] In some embodiments, the second mammalian species is a pig, and in some embodiments, the pig is a miniature pig. In some embodiments, the first mammalian species is a primate. In some embodiments, the recipient mammal is a human.

[0031] In some embodiments, the thymus tissue from the second mammalian species is fetal thymus tissue. In some embodiments, the thymus tissue from the second mammalian species is neonatal thymus tissue.

[0032] In some embodiments, the thymic epithelial cells are obtained from a thymus from a first mammalian species. In some embodiments, the thymic epithelial cells from a first mammalian species are obtained from fetal thymic tissue. In some embodiments, the thymic epithelial cells from a first mammalian species are obtained from neonatal thymic tissue. In some embodiments, the thymic epithelial cells are generated from induced pluripotent stem cells (iPSCs) from a first mammalian species. In some embodiments, the thymic epithelial cells are generated from embryonic stem cells that share HLA alleles with the first mammalian species. In some embodiments, the embryonic stem cells are genetically engineered to share HLA alleles with the first mammalian species.

[0033] The present disclosure also provides an isolated hybrid thymic tissue comprising thymic epithelial cells from a first mammalian species and thymic tissue from a second mammalian species, and a method of making the hybrid thymic tissue, comprising: (i) treating thymus tissue from a second mammalian species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from the first mammalian species into the 2DG-treated thymic tissue; The present invention provides a method comprising:

[0034] In some embodiments, thymic epithelial cells from a first mammalian species are suspended in Matrigel before being injected into the 2DG-treated thymic tissue.

[0035] For the purpose of illustrating the invention, there is shown in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0036] [Figure 1]Generation of human / pig hybrid thymus. At 12–20 weeks post-transplant, humanized mice generated with hybrid pig / human thymus and human CD34+ cells were euthanized, and the transplanted thymus was removed, sectioned, and stained to detect human TECs using two-photon confocal microscopy. Figures 1A, 1B, and 1C show images of transplanted pig thymuses that were uninjected (Figure 1A), injected with human fetal thymic stromal cells (20 weeks gestational age) (Figure 1B), and injected with pediatric human thymic stromal cells (derived from a 4-month-old thymus) (Figure 1C). Arrows in B and C point to CK14+HLA-DR+ cells, representing human TECs within the pig thymus. A quantitative analysis of the entire section shown in Figures 1A–1C is shown in Figure 1D. Additional controls from human fetal and pediatric thymuses and pig thymuses were also included in the quantitative analysis. Analysis was performed using ImarisColoc software, which allows for the calculation of the localization of CK14+HLA-DR+ cells within all CK14+ cells in the entire thymic section. The numbers at the top of the bars represent the percentage of CK14+HLA-DR+ cells among CK14+ cells. Figures 1E and 1F show representative images of human thymic mesenchymal cells (TMCs) (Figure 1E) and TECs (Figure 1F) expanded in 3-D Matrigel cultures for 3 weeks from huCD45-depleted human thymocytes after digestion of the thymus of a 17-year-old child with Liberase. Figures 1G and 1H show representative flow cytometry characteristics of expanded TMCs and TECs, respectively. CD105-CD326+ cells are considered TECs, while CD105+CD326- cells are considered TMCs. [Figure 2] CK14+HLA-DR+ cells were detected in hybrid thymuses generated by injecting human thymic stromal cells (TECs) (derived from fetal thymus) into pig thymuses. Figure 2A: No human TEC injection; Figure 2B: Human TEC injection; Figure 2C: 2-DG-treated pig thymus + human fetal TEC injection. [Figure 3]Figure 3 shows results demonstrating the development of a method for use in generating hybrid thymi. Figure 3A shows flow cytometry results indicating the number of cells released after injection of cells into fetal porcine thymus fragments. PBMC cells were resuspended in Matrigel to prevent leakage from the porcine thymus after injection. Three different methods were used to inject cells into thawed fetal porcine thymus fragments. Method A: The fragments were placed in the wells of a V-bottom 96-well plate and injected using a Hamilton syringe. Method B: Injection using PE50 tubing. Method C: Injection using a Hamilton syringe while holding the fragments outside the wells with forceps until the Matrigel solidified. The number of released cells was determined by flow cytometry, and injected and CFSE-stained PBMCs were tracked. Figures 3B and 3C show the results of various reagents for ex vivo depletion of thymocytes in fetal porcine thymus fragments. Figure 3B shows graphs of the total viable cell counts (top panel) and the percentage of viable cells (bottom panel) for cells treated with each reagent. Figure 3C shows graphs of the ratio of single-positive (SP) CD4 (SP-CD4) or SP-CD8 cells over the remaining viable double-positive CD4 and CD8 cells (DP) or double-negative (DN) cells containing stromal cells used as readout. Treatment with 100 nM 2DG for 12 hours resulted in the lowest ratios and was therefore the best strategy for depleting thymocytes while preserving stromal cells. [Figure 4] Figure 4 shows the experimental design for preparing hybrid thymus and transplanting the hybrid thymus into recipient mice. [Figure 5]Figure 5 shows graphs of the level of human immune cell reconstitution after hybrid thymic transplantation of fetal pig thymi that were treated with 2DG and injected with human thymic stromal cells (represented by circles on the graph), not treated with 2DG and injected with human thymic stromal cells (represented by squares on the graph), or not treated with 2DG and not injected with human thymic stromal cells (represented by triangles on the graph). Figure 5A shows the percentage of hCD45+ cells among leukocytes. Figure 5B shows the percentage of CD3+ cells among hCD45+ cells. Figure 5C shows the number of hCD45+ cells per μL of blood. Figure 5D shows the number of hCD3+ cells per μL of blood. Figure 5E shows the percentage of CD4+ cells among hCD3+ cells. Figure 5F shows the number of hCD4+ cells per μL of blood. Figure 5G shows the percentage of CD19+ cells among hCD45+ cells. Figure 5H shows the percentage of CD14+ cells among hCD45+. Figure 5I shows the number of hCD19+ cells per μl of blood. Figure 5J shows the number of hCD14+ cells per μl of blood. Figure 5K shows the percentage of naive cells among hCD3+. Figure 5L shows the percentage of effector memory cells among hCD3+. [Figure 6] Figure 6 shows images of the detection of injected human TECs mixed with porcine TECs within the transplanted thymus. An uninjected porcine thymus transplanted into a humanized mouse is shown on the left, and a hybrid thymus transplanted into a humanized mouse is shown on the top right. [Figure 7] Figure 7 shows graphs of in vitro T cell proliferation results suggesting that peripheral T cells from mice bearing hybrid thymi are partially tolerant to the human TEC donor. Figure 7A shows the T cell proliferation response of mice bearing various transplanted thymi to pig dendritic cells. Figure 7B shows the T cell proliferation response of mice bearing various transplanted thymi to human pig dendritic cells. Circles represent fetal pig thymi that were not injected with human thymic stromal cells, squares represent fetal pig thymi that were injected with human thymic stromal cells but not treated with 2-DG, and triangles represent fetal pig thymi that were injected with human thymic stromal cells that were treated with 2-DG. [Figure 8]Figure 8 shows increased responses to human tissue-restricted antigens (TRAs) (MART-1, NYESO1, and islet antigen IA-2) in human T cells developing in the pig thymus (SW / HU mice) compared with T cells developing in the human thymus (HU / HU mice). Figure 8A is a schematic diagram of the mouse model. Figure 8B is a graph showing the proliferative response of human peripheral T cells from mice (18 weeks post-transplant) to human TRAs (IA-2, MART-1, and NYESO1) presented by human HSC donor DCs. [Figure 9]Figure 9 shows poorer survival of human Tregs and CD8 T cells developed in pig thymus (SW / HU mice) compared to those developed in human thymus (HU / HU mice). Figure 9 is a graph showing various cells in transplanted thymocytes in HU / HU mice compared to SW / HU mice, and various cells in the spleen / lymph nodes in HU / HU mice compared to SW / HU mice. HU / HU mouse numbers are indicated as circles, and SW / HU mouse numbers are indicated as squares. Figure 9A shows the total number of thymocytes in the transplanted thymus. Figure 9B shows the proportion of thymocyte subsets in the transplanted thymus. Figure 9C shows the proportion of Tregs within SP-CD4+ in the transplanted thymus. Figure 9D shows the proportion of Ki67+ cells in the transplanted thymus. Figure 9E shows the proportion of CD45RO+ cells in the transplanted thymus. Figure 9F shows the proportion of CTLA-4+ cells in the transplanted thymus. Figure 9G shows the total cell counts in the spleen and lymph nodes (LN) of each mouse subset. Figure 9H shows the percentage of hCD45+ cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9I shows the percentage of T cells among hCD45+ cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9J shows the percentage of CD4 and CD8 cells among T cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9K shows the percentage of Treg cells among CD4+ cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9L shows the percentage of naive cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9M shows the percentage of EM cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9N shows the percentage of HLA-DR+ cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9O shows the percentage of Ki67+ cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9P shows the percentage of hCD45RO+ cells in the spleen and lymph nodes (LN) of each mouse subset. Figure 9Q shows the percentage of CTLA-4+ cells in the spleen and lymph nodes (LN) of each mouse subset. [Figure 10]Figure 10 shows the long-term (>20 weeks) persistence of human TECs in "hybrid thymus." Figure 10A shows images of transplanted pig thymuses, including those not injected with human TECs (SW / THY, top left panel), pig thymuses injected with human fetal TECs (SW / fetal hu-TES THY, top right panel), pig thymuses injected with human pediatric TECs (SW / pediatric hu-TEC THY, bottom left panel), and pig thymuses injected with human hPSCs (SW / hPSC-TEC THY, bottom right panel). Figure 10B shows representative flow cytometry staining of gated CD45-negative cells in digested stroma from various long-term thymic transplants, demonstrating the presence of EPCAM+, CD105-negative hu-TECs only in human thymuses (top right) and SW transplants injected with hPSC-TEC precursors (bottom left panel), but not in uninjected SW THY transplants (top left). Figure 10C is a graph of quantification of the percentage of CD45-HLA-ABC+EpCAM+ (injected human TECs) from multiple mice receiving SW thymus injected with human ES-TECs relative to uninjected SW thymus. [Figure 11]Figure 11 shows that injection of hES-TECs into pig thymi enhances T cell development and promotes peripheral CD4+ and CD8+ T cell expansion. Pig thymi injected with 1–2 × 105 hES-TECs (green bars) or uninjected (clear bars) were transplanted under the renal capsule of thymectomized NSG mice that had been intravenously injected with 2 × 105 human fetal liver-derived CD34+ cells. Splenocytes and thymocytes from the thymic grafts were analyzed by flow cytometry at 18–22 weeks post-transplant. Figure 11A shows the absolute number of human CD3+ T cells in each group of mice. Figure 11B shows the absolute number of human CD8+ T cells in each group of mice. Figure 11C shows the absolute number of human CD4+ T cells in each group of mice. Figure 11D is a graph of the absolute number of recent thymic emigrant CD31+CD4+ naive cells, defined as CD45RA+CCR7+ cells, in splenic mononuclear cells in each group of mice. Figure 11E is a graph of the number of indicated cells in thymocytes in each group of mice stained for HuCD45, CD19, CD14, CD4, CD8, CD45RA, and CD45RO expression. Thymocytes were gated as huCD45+CD19-CD14- cells. Absolute numbers of thymocytes from half of the thymic graft, gated as total human CD45 cells, double-positive CD4+CD8+, single-positive CD4+CD8-, and CD4-CD8+, are shown. SP CD4 and CD8 cells were further subgated to immature CD45RO+ cells compared to more mature CD45RA+ thymocytes. Means + SEM are shown for pig thymi injected with hES-TECs (n = 6, squares) and pig thymi alone (n = 5, triangles), representing two independent experiments. Thymic grafts containing fewer than 6 x 10 cells (n = 1 each from SwTHY and SwTHY + TEC) were excluded from the analysis. p values ​​were determined using the Mann-Whitney test comparing the SwTHY hES-TEC-injected group with the SwTHY-only-injected group, with p < 0.05 considered significant. *p < 0.05, +p = 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention Abbreviation SW-Pig HU-Human TEC-thymic epithelial cells TMC-thymic mesenchymal cells WBC-white blood cells DP-double positive cells (both CD4+ and CD8+) SP - single positive cells (either CD4+ or CD8+) treg - regulatory T cells LN - Lymph Node TRA-tissue-specific autoantigen 2DG - 2D glucose MACS-Magnetic Activated Cell Sorting HSC - human hematopoietic cells

[0038] The present disclosure provides a method for generating a human / pig hybrid thymus to achieve immune tolerance to pig antigens with optimal immune function of the generated human T cell repertoire. This method overcomes the limitations encountered when using a single pig thymus by improving human T cell function and self-tolerance in the pig thymus while developing tolerance to pig xenografts.

[0039] This method generates a hybrid pig-human thymus containing patient-specific thymic epithelial cells (TECs). Patient-specific TECs can be obtained directly from the patient's thymus, generated from patient-specific induced pluripotent stem cells, or generated from embryonic stem cells that naturally or artificially share human leukocyte antigen (HLA) alleles with the patient. Patient-specific TECs can participate in positive selection, resulting in T cells that more readily recognize foreign antigens presented by recipient HLA molecules in the periphery. Furthermore, because many tissue-specific antigens (TSAs) differ between humans and pigs, adding human TECs to generate human TSAs in the thymus helps ensure protection from autoimmunity. Thus, using a hybrid thymus instead of a pig thymus can improve the function and self-tolerance of the generated human T cell repertoire and enable the induction of xenograft tolerance.

[0040] The hybrid thymus / thymus tissue (e.g., hybrid pig-human thymus / thymus tissue) of the present invention can also be used for immune reconstitution in patients who lack adequate thymic function or have T cell immunodeficiency (e.g., senescent thymus in adults). Applications of hybrid thymus / thymus tissue (e.g., hybrid pig-human thymus / thymus tissue) include pharmacology and drug screening, medical xenograft tolerance testing and preparation, and xenogeneic models for tolerance induction to therapeutic molecules that can cause immunogenicity (e.g., mAbs) used during transplantation or immune disorder treatment.

[0041] As used herein, hybrid thymic tissue refers to thymic tissue from a donor mammal of a second species, which contains thymic epithelial cells from a recipient mammal of a first species.

[0042] In one embodiment, a hybrid thymus / thymus tissue is constructed in which pig thymus / thymus tissue (e.g., fetal thymus / thymus tissue) containing human (e.g., patient-specific) thymic epithelial cells (TECs) is obtained from human thymus / thymus tissue (e.g., patient thymus / thymus tissue). In another embodiment, a hybrid thymus / thymus tissue is constructed in which pig thymus / thymus tissue (e.g., fetal thymus / thymus tissue) containing human (e.g., patient-specific) TECs is generated from human (e.g., patient-specific) induced pluripotent stem cells. In yet another embodiment, a hybrid thymus / thymus tissue is constructed in which pig thymus / thymus tissue (e.g., fetal thymus / thymus tissue) containing human TECs is generated from embryonic stem cells that naturally share HLA alleles with the patient or have been engineered to do so.

[0043] The inclusion of human TECs into pig thymus grafts can have functional effects on antigen recognition.

[0044] The present disclosure provides a method for inducing tolerance in a recipient mammal of a first species to a graft obtained from a donor mammal of a second species. The method may include the following steps: (a) introducing hybrid thymic tissue from the second species, the hybrid thymic tissue comprising thymic epithelial cells from the first species, into the recipient mammal; and (b) transplanting the graft from the donor mammal into the recipient mammal. The donor may be a pig, including a minipig. The recipient may be a human.

[0045] The present disclosure also provides methods for generating primate-pig hybrid thymus / thymic tissue to achieve immune tolerance to porcine antigens with optimal immune function of the generated primate T cell repertoire. One embodiment of the present disclosure provides hybrid thymus / thymic tissue in pigs with baboons.

[0046] The hybrid thymus / thymic tissue can be transplanted primarily as vascularized thymic lobes or as composite thymus-kidney grafts.

[0047] The hybrid thymus / thymus tissue can be transplanted intramuscularly into a recipient. The hybrid thymus / thymus tissue can be transplanted either into the quadriceps muscle alone or into additional transplantation sites (e.g., the kidney capsule and omentum) in the recipient. Wu et al., Xenogeneic Thymus Transplantation in a Pig-to-Baboon Model, Transplantation, 2003, 75(3):282-291.

[0048] The recipient of a xenotransplant is a mammal of a first mammalian species. The donor of a xenotransplant refers to a mammal of a second mammalian species. The donor mammal is the donor of cells, tissues, and / or organs for the xenotransplant.

[0049] The present disclosure provides a method of inducing tolerance in a recipient mammal of a first species to a graft obtained from a donor mammal of a second species, the method comprising the steps of (a) introducing into the recipient mammal hybrid thymic tissue from the second species, the hybrid thymic tissue comprising thymic epithelial cells from the first species, and (b) transplanting the graft from the donor mammal in the recipient mammal. Step (a) may be performed before or simultaneously with step (b).

[0050] The present disclosure also provides a method of restoring or inducing immunocompetence in a recipient mammal of a first species, comprising introducing into the recipient mammal thymic tissue from a donor mammal of a second species, the hybrid thymic tissue comprising thymic epithelial cells from the first species.

[0051] Also encompassed by the present disclosure is a method for restoring or promoting thymic dependency of T cell precursors that develop into mature functional T cells in a recipient mammal of a first species, the method comprising introducing into the recipient mammal thymic tissue from a donor mammal of a second species, hybrid thymic tissue comprising thymic epithelial cells from the first species.

[0052] In one embodiment, thymic function is essentially absent in the recipient mammal prior to the introduction of the hybrid thymic tissue. In another embodiment, the recipient mammal is thymectomized prior to the introduction of the hybrid thymic tissue. In yet another embodiment, the recipient mammal has an immune disorder.

[0053] The second species may be a pig, such as a miniature pig.

[0054] The first species can be a primate, such as a non-human primate or a human.

[0055] In one embodiment, the recipient mammal is a human and the donor mammal is a minipig.

[0056] The thymus tissue from the second species can be fetal thymus tissue or neonatal thymus tissue.

[0057] The thymic epithelial cells from the first species can be obtained from the thymus of the recipient mammal. The thymic epithelial cells from the first species can be produced from induced pluripotent stem cells (iPSCs) of the recipient mammal. The thymic epithelial cells from the first species can be produced from embryonic stem cells that share HLA alleles with the recipient mammal. For example, the embryonic stem cells can naturally share HLA alleles with the recipient mammal or be genetically engineered to share HLA alleles with the recipient mammal.

[0058] In one embodiment, the hybrid thymic tissue is transplanted into a recipient mammal. For example, the hybrid thymic tissue can be transplanted primarily as vascularized thymic lobes or as a composite thymus-kidney graft.

[0059] Hybrid thymic tissue can be created by introducing thymic epithelial cells from a first species into thymic tissue from a second species. Hybrid thymic tissue can be created by injecting thymic epithelial cells from a first species into thymic tissue from a second species.

[0060] Hybrid thymic tissue can be produced by a method comprising the following steps: (i) treating thymic tissue from a second species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from a first species into the 2DG-treated thymic tissue. In step (ii), the thymic epithelial cells can be suspended in a biomaterial such as Matrigel before being injected into the 2DG-treated thymic tissue.

[0061] The thymic epithelial cells can be suspended in a biomaterial (eg, Matrigel) before being injected into the thymic tissue from the second species.

[0062] In some embodiments, thymic epithelial cells from a first species can be combined with (e.g., suspended in) a biomaterial before being introduced into thymic tissue from a second species. The biomaterial can be a sol-gel, a protein-containing hydrogel, Matrigel, an artificially constructed scaffold with cells, or a combination thereof. Non-limiting examples of biomaterials can include polyethyleneimine and dextran sulfate, poly(vinylsiloxane) copolymer polyethyleneimine, phosphorylcholine, poly(ethylene glycol), poly(lactic-co-glycolic acid), poly(lactic acid), polyhydroxyvaleric acid and copolymers, polyhydroxybutyrate and copolymers, polydioxanone, polyanhydrides, poly(amino acids), poly(orthoesters), polyesters, collagen, gelatin, cellulose polymers, chitosan, alginate, fibronectin, extracellular matrix proteins, vinculin, agar, agarose, hyaluronic acid, Matrigel, and combinations thereof.

[0063] The method may further comprise administering hematopoietic stem cells (HSCs) to the recipient mammal.

[0064] The graft may comprise cells, tissues, or organs. In one embodiment, the graft comprises hematopoietic stem cells. In another embodiment, the graft comprises bone marrow. In yet another embodiment, the graft comprises heart, kidney, liver, pancreas, lung, intestine, skin, small intestine, trachea, cornea, or a combination thereof.

[0065] The present disclosure provides isolated hybrid thymic tissue comprising thymic epithelial cells from a first mammalian species and thymic tissue from a second mammalian species.

[0066] Alternative approaches to achieving central T-cell tolerance of highly dissimilar xenogeneic donors have been developed, including transplantation of porcine thymus into immunocompetent, T-cell-depleted, and thymectomized recipients. These studies, initiated in mice, demonstrated remarkable and specific in vitro non-responsiveness and prolonged survival of donor-specific skin grafts (Lee LA, Gritsch HA, Sergio JJ, et al., Specific tolerance across a discordant xenogeneic transplantation barrier. Proc Natl Acad Sci USA. 1994;91:10864-10867. Zhao Y, Swenson K, Sergio JJ, Arn JS, Sachs DH, Sykes M. Skin graft tolerance across a discordant xenogeneic barrier. Nature Med. 1996;2:1211-1216). The mouse model has enabled extensive investigation of the mechanisms of tolerance and immune function conferred by T cell reconstitution in xenogeneic thymic grafts. Intrathymic clonal deletion is a major mechanism that tolerizes newly developed thymocytes to xenogeneic donors and recipients (Zhao Y, Sergio JJ, Swenson KA, Arn JS, Sachs DH, Sykes M. Positive and negative selection of functional mouse CD4 cells by porcine MHC in pig thymus grafts. J Immunol. 1997;159:2100-2107. Zhao Y, Rodriguez-Barbosa JI, Shimizu A, Swenson K, Sachs DH, Sykes M.).Despite efficient intrathymic negative selection of host-reactive T cells, autoimmune disease can develop in athymic mice transplanted with pig thymus: Evidence for failure of regulatory mechanisms suppressing autoimmunity (Transplantation. 2002;75:1832-1840). Additional studies have implicated Tregs developing in pig thymus grafts in suppressing residual mouse anti-pig responses. Zhao et al.: The induction of specific pig skin graft tolerance by grafting with neonatal pig thymus in thymectomized mice. Transplantation. 2000; 69:1447-1451. Rodriguez-Barbosa et al.: Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of pre-existing xenoreactive T cells. Transplantation. 2001;72:1223-1231. Using T cell receptor (TCR) transgenic recipient mice with different MHC haplotypes and TCRs for which mouse MHC alleles had previously been identified, it was demonstrated that positive selection in pig thymus grafts was mediated exclusively by pig thymic MHC, with no contribution from mouse hematopoietic cells, whereas negative selection was mediated by both pig and mouse MHC, consistent with the presence of class II MHC+ APCs from both species in the donor pig thymus grafts.Zhao Y, Rodriguez-Barbosa JI, Zhao G, Shaffer J, Arn JS, Sykes M. Maturation and function of mouse T cells with a transgeneic TCR positively selected by highly disparate xenogeneic porcine MHC. Cell Mol Biol. 2000;47:217-228. Zhao Y, Swenson K, Sergio JJ, Sykes M. Pig MHC mediates positive selection of mouse CD4+ T cells with a mouse MHC-restricted TCR in pig thymus grafts. J Immunol. 1998;161:1320-1326. Despite the lack of mouse MHC involvement in positive selection and the complete MHC disparity between the pig thymus and the mouse recipient, these T cells were able to respond to immunization with protein antigens presented by mouse MHC molecules and, most importantly, protect mice from opportunistic pathogens whose clearance is dependent on CD4+ T cells ( Zhao et al., Immune restoration by fetal pig thymus grafts in T cell-depleted, thymectomized mice , J Immunol. 1997;158:1641-1649 ). These results are interpreted as demonstrating that when a diverse T cell repertoire is selected in a xenogeneic thymus graft, sufficient cross-reactivity for recognition of foreign antigens on the recipient's MHC can occur.

[0067] The pig thymus transplantation method for tolerance has been extended to a humanized mouse model to provide proof of principle that human T cells can develop normally and be centrally tolerized to pig xenoantigens in the pig thymus graft. Nikolic B, Gardner JP, Scadden DT, Arn JS, Sachs DH, Sykes M. Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. J Immunol. 1999;162:3402-3407. Kalscheuer HO,T.; Dahmani,A.; Li,H.; Holzl,M.; Yamada,K.; Sykes,M. Xenograft tolerance and immune function of human T cell developing in pig thymus xenografts. J Immunology.2014;192(7):3442-3450. Both thymic and peripheral human T cells developing in pig thymus grafts exhibit specific non-responsiveness to the donor pig, with intact responses to third-group pigs and allogeneic humans in mixed lymphocyte reactions (MLRs). These T cells also exhibit non-responsiveness to the human hematopoietic stem cell (HSC) donor and mouse recipient in MLRs, both of which reflect the contribution of human donor APCs and mouse APCs to the negative selection detected in thymic xenografts. Kalscheuer et al., A model for personalized in vivo analysis of human immune responsiveness. Science Translational Medicine. 2012;4(125):125ra130. Importantly, donor-specific skin graft tolerance is observed for human T cells developing in pig thymus grafts.

[0068] Based on the results of mouse models, the thymus xenotransplantation technique has been extended to the large animal species combination of pig and baboon. Early studies using pig thymus fragments placed under the baboon kidney capsule demonstrated some T cell recovery, donor-specific hyporesponsiveness in vitro, and prolonged survival of donor skin grafts compared with controls. However, the amount of transplanted, vascularized pig thymus tissue was very limited. Wu et al., Xenogeneic thymus transplantation in a pig-to-baboon model, Transplantation. 2003;75(3):282-291. To achieve more robust thymic function, and in light of the mouse data described above, it was expected that donor-specific Tregs developed in the pig thymus would be required to suppress pre-existing T cells that are not depleted by transplant conditioning. Subsequent studies primarily utilized vascularized pig thymuses, which have already been shown to be effective in inducing tolerance in an allogeneic pig kidney transplantation model. Yamada K, Shimizu A, Utsugi R, et al. Thymus transplantation in miniature swine. II. Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients. J Immunol.2000;164:3079-3086. Thymuses were transplanted as part of a composite "thymokidney" graft prepared several months earlier in donor pigs by placing autologous thymus fragments under the pig's renal capsule, or by direct vascular anastomosis of pig thymic lobes in baboons. Yamada K, Yazawa K, Shimizu A, et al.Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nature medicine. 2005;11(1):32-34. Both approaches resulted in the long-term survival of GalT knockout pig kidneys in baboons for the first time. Tasaki et al., Rituximab treatment prevents the early development of proteinuria following pig-to-baboon xeno-kidney transplantation. Journal of the American Society of Nephrology: JASN. 2014;25(4):737-744. Survival of animals receiving this treatment is limited by thrombotic complications of anti-CD40L and by proteinuria due to minimal change disease-like glomerulopathy, which can be avoided by the use of nonthrombotic anti-CD40 and by administering rituximab and CTLA 4Ig, respectively. Yamada et al., Xenotransplantation: Where Are We with Potential Kidney Recipients? Recent Progress and Potential Future Clinical Trials. Curr Transplant Rep. 2017;4(2):101-109.

[0069] Baboons receiving pig thymic-kidney grafts showed evidence of de novo recipient (baboon) thymic lymphopoiesis in the pig thymus graft, the appearance of recent thymic emigration in the periphery and donor-specific nonresponsiveness in Elispot and MLR assays, and a decline in non-Gal natural antibodies. (Tanabe et al., "Role of Intrinsic (Graft) Versus Extrinsic (Host) Factors in the Growth of Transplanted Organs Following Allogeneic and Xenogeneic Transplantation," Am J Transplant. 2017 Jul;17(7):1778-1790. Although the latter may reflect absorption by the pig kidney, minimal IgM binding was detected in these xenografts, and there was no complement fixation or significant pathology. Therefore, the results obtained in this model demonstrate the potential of composite thymic-kidney xenografts to induce tolerance in primates.

[0070] Limitations of generating a human T cell repertoire in a xenogeneic pig thymus include preferential recognition of microbial antigens on pig MHC, which is useful for protecting the graft but does not optimize protection against microbial pathogens infecting the host, poorly selects for conventional T cells, and fails to actively select for Tregs that recognize human tissue-specific autoantigens (TRAs). Indeed, studies in humanized mice have shown diminished responses to peptides presented by human APCs following immunization when human T cells are developed in pigs rather than in human thymus grafts.

[0071] Approaches to overcome this limitation include the creation of a "hybrid thymus" in which recipient thymic epithelial cells, either obtained from a thymectomy specimen or generated from stem cells, are injected into pig thymic tissue. A hybrid thymus from a postnatal thymic donor is created, and the hybrid thymus promotes tolerance to human TRAs among human T cells.

[0072] Pig thymus grafts have been shown to support the development of normal and diverse murine or human T cell repertoires, and these T cells have been shown to be specifically tolerant to the xenogeneic pig donor. However, recognition of foreign antigens presented by the recipient's HLA molecules in the periphery is suboptimal. Therefore, immune function may be suboptimal. As shown herein, this can be overcome by providing recipient TECs in a pig-human hybrid thymus graft, as these TECs participate in positive selection, resulting in T cells that can more easily recognize foreign antigens presented by recipient HLA molecules in the periphery. In the case of pig thymus grafts, the survival, homeostasis, and function of T cells in the absence of their "positive selection" ligands in the periphery are suboptimal. Positive selection ligands are MHC / peptide complexes on TECs that rescue thymocytes from programmed cell death when the thymocytes possess low-affinity T cell receptors that recognize the complex. Providing recipient TECs in a pig-human hybrid thymus allows for the positive selection of T cells bearing the same ligands on recipient cells in the periphery, conferring normal survival, homeostasis, and function. In the case of pig thymus grafts, TECs produce antigens that are otherwise expressed only in highly specific surrounding tissues (i.e., tissue-specific antigens, or TSAs). Two important consequences of this expression of TSAs by TECs are: a) clonal deletion of thymocytes that strongly recognize these autoreactive T cells, depleting them from the repertoire; and b) positive selection of regulatory T cells that recognize them, adding a safety net to prevent peripheral autoimmunity. Because many TSAs differ between humans and pigs, adding human TECs to generate human TSAs in pig-human hybrid thymus grafts would help ensure protection from autoimmunity.

[0073] In summary, by using a hybrid thymus instead of a single pig thymus, it is possible to improve the function and self-tolerance of the human T cell repertoire generated in the pig thymus, while developing tolerance to the pig.

[0074] In one embodiment, the recipient is thymectomized. In another embodiment, the recipient is not thymectomized. In yet another embodiment, the recipient has a slow rate of thymus formation due to age. In yet another embodiment, the recipient has a senescent thymus.

[0075] Thymic xenotransplantation using the hybrid thymus / thymus tissue of the present invention may or may not be combined with mixed chimerism induction. For example, when thymic xenotransplantation is combined with durable mixed pig-human chimerism induction, both pig and human APCs are present in the native human thymus and pig thymus xenograft, ensuring lifelong negative selection of thymocytes that recognize either pig or human antigens expressed on hematopoietic cells. Furthermore, conventional T cells that recognize pig or human TRAs are deleted in the thymus of the related species, and evasion of deletion due to development in the thymus of the opposite species is appropriately suppressed by TRA-specific Tregs that develop in the other thymus. Mixed pig chimerism ensures tolerance of natural antibodies that recognize unknown xenogeneic targets, and NK cells are similarly tolerized.

[0076] Xenotransplantation lends itself more readily to tolerance induction than allotransplantation from deceased donors, as the ability to perform xenotransplantation selectively allows for the application of tolerance protocols (e.g., mixed chimerism induction) prior to organ xenografting. In one embodiment, the method involves first tolerizing the recipient's immune system, ensuring that tolerance is achieved, and then performing the organ transplant without immunosuppression or with a shortened course of immunosuppression.

[0077] The present disclosure provides a method for inducing tolerance in a recipient mammal of a first species (e.g., a primate such as a human) to a graft obtained from a mammal of a second species, e.g., a pig. The method involves introducing a hybrid thymus / thymic tissue into the recipient mammal prior to or concurrently with transplantation of the graft, and (optionally) transplanting the graft into the recipient. The hybrid thymus / thymic tissue prepares the recipient for the subsequent transplant by inducing immune tolerance at the T cell level.

[0078] The present disclosure provides a method for inducing xenograft tolerance in a recipient, the method comprising introducing hybrid thymus / thymic tissue into the recipient.

[0079] In one embodiment, host T cells from an athymic, T cell-depleted recipient transplanted with hybrid thymus / thymic tissue are able to mature in the hybrid thymus / thymic tissue. The host T cells that mature in the transplanted hybrid thymus / thymic tissue are immunocompetent.

[0080] The present disclosure provides methods for restoring or inducing immunocompetence (or restoring or promoting thymus dependency of T cell progenitor cells to mature or develop into functional mature T cells) in a host or recipient, e.g., a primate host or recipient, that is capable of producing T cell precursors but is unable to produce sufficient numbers of mature, functional T cells for a normal immune response due to thymic insufficiency. The methods include introducing hybrid thymus / thymus tissue into the recipient such that host T cells can mature in the transplanted hybrid thymus / thymus tissue.

[0081] In one embodiment, the recipient / host is a primate, eg, a human, and the donor is a pig, eg, a minipig.

[0082] The method can include other steps that facilitate acceptance of the hybrid thymus / thymus tissue or otherwise optimize the method. In certain embodiments, liver or spleen tissue, such as fetal or neonatal liver or spleen tissue, is transplanted with the thymus tissue, and donor hematopoietic cells, e.g., umbilical cord blood stem cells or fetal or neonatal liver or spleen cells, are administered to the recipient, e.g., a suspension of fetal liver cells is administered intraperitoneally or intravenously. The recipient can be thymectomized, such as before or at the time the hybrid thymus / thymus tissue is introduced.

[0083] In certain embodiments, the methods include depleting, inactivating, or inhibiting natural killer (NK) cells to prevent NK-mediated rejection of the thymic tissue (preferably, before or at the time of introducing thymic tissue into the recipient), e.g., by introducing into the recipient an antibody capable of binding to the recipient's NK cells; depleting, inactivating, or inhibiting host T cell function (preferably, before or at the time of introducing thymic tissue into the recipient), e.g., by introducing into the recipient an antibody capable of binding to the recipient's T cells; or depleting, inactivating, or inhibiting host CD4+ cell function (preferably, before or at the time of introducing thymic tissue into the recipient), e.g., by introducing into the recipient an antibody capable of binding to the recipient's CD4 or CD4+ cells.

[0084] Some embodiments include creating hematopoietic space by, for example, administering to the recipient mammal low doses of irradiation, e.g., about 100 to 400 rads, total body irradiation, administration of a myelosuppressant, or administration of hematopoietic stem cells with inactivating or depleting antibodies (preferably prior to transplantation of thymic tissue or hematopoietic stem cells) to deplete or partially deplete the recipient's bone marrow.

[0085] Some embodiments include inactivating thymic T cells (preferably prior to transplantation of thymic tissue or hematopoietic stem cells) by one or more of irradiating the host with, for example, about 700 rads of thymic irradiation, administering to the recipient one or more doses of an anti-T cell antibody, e.g., an anti-CD4 and / or anti-CD8 monoclonal antibody, or administering to the recipient an immunosuppressant for a short period of time.

[0086] Some embodiments include depleting or otherwise inactivating natural antibodies by one or more of the following: administration of a drug that depletes or inactivates natural antibodies, e.g., deoxyspergualin, administration of an anti-IgM antibody, or adsorption of natural antibodies from the host's blood, e.g., by contacting the host's blood with donor antigens, e.g., by hemoperfusion of a donor organ, e.g., a kidney or liver, from the donor species.

[0087] Other methods may be combined with the methods disclosed herein to promote recipient acceptance of the graft. For example, tolerance to thymic tissue can also be induced by inserting a nucleic acid expressing a donor antigen, e.g., a donor MHC gene, into recipient cells, e.g., hematopoietic stem cells, and introducing the genetically engineered cells into the recipient. For example, human recipient stem cells can be engineered to express pig MHC genes, e.g., pig class I or class II MHC genes, or both class I and class II genes, and the cells are transplanted into a human recipient that receives the hybrid thymic tissue. When inserted into a recipient primate, e.g., a human, expression of the donor MHC gene can result in tolerance to subsequent exposure to the donor antigen, thereby inducing tolerance to thymic tissue.

[0088] For example, methods of inducing tolerance by hematopoietic stem cell transplantation can be combined with the methods disclosed herein.

[0089] Other methods of inducing tolerance may be used to promote thymic tissue acceptance. For example, suppression of helper T cells, which can be induced by short-term administration of high doses of immunosuppressants, such as cyclosporine, has been found to induce tolerance. In these methods, helper T cells are suppressed for a relatively short period immediately after transplantation of the graft, and do not require or include chronic immunosuppression.

[0090] Other methods of promoting tolerance or acceptance of donor tissue, for example, by altering the level of cytokine activity or inhibiting graft-versus-recipient disease, may also be used in combination with the methods of the present invention.

[0091] In another aspect, the present disclosure provides a method for reducing or inhibiting T cell activity, preferably thymic or lymph node T cell activity, in a recipient mammal, e.g., a primate, e.g., a human, receiving a graft from a donor mammal. The method comprises inducing tolerance to the graft and administering an immunosuppressant, e.g., cyclosporine, to the recipient for a short period of time sufficient to inactivate T cells, preferably thymic or lymph node T cells.

[0092] As used herein, "thymic deficiency" refers to a condition in which the ability of an individual's thymus to support T cell maturation is impaired compared to a normal individual. Athymic conditions include those in which the thymus or thymic function is essentially absent.

[0093] As used herein, "tolerance" refers to the inhibition or diminution of a transplant recipient's ability to mount an immune response, e.g., to donor antigens, that would otherwise occur, e.g., in response to the introduction of non-self MHC antigens to the recipient. Tolerance can include humoral, cellular, or both humoral and cellular responses. The concept of tolerance includes both complete and partial tolerance. In other words, as used herein, tolerance includes any degree of inhibition of a transplant recipient's ability to mount an immune response, e.g., to donor antigens.

[0094] As used herein, "hematopoietic stem cells" refer to cells that can develop into mature myeloid and / or lymphoid cells. Preferably, hematopoietic stem cells allow for long-term repopulation of myeloid and / or lymphoid lineages. Stem cells derived from the umbilical cord blood of the recipient or donor can be used in the methods of the present disclosure.

[0095] As used herein, "minipigs" refers to fully or partially inbred minipigs.

[0096] As used herein, "graft" refers to a body part, organ, tissue, cell, or portion thereof.

[0097] As used herein, "stromal tissue" refers to the supporting tissue or matrix of an organ, as distinguished from its functional elements or substance.

[0098] As used herein, restoring, inducing, or promoting immune competence means one or both of: (1) increasing the number of mature, functional T cells in the recipient (above that seen in the absence of treatment by the methods of the present disclosure), either by increasing the number of recipient mature, functional T cells or by providing mature, functional donor T cells in the recipient; or (2) improving the immune responsiveness of the recipient, e.g., as measured by the ability to mount a cutaneous response to a recall antigen, or improving the responsiveness of the recipient's T cells, e.g., by an improved proliferative response to an antigen, e.g., an improved response to a tetanus antigen or an alloantigen, as measured, e.g., by in vitro testing.

[0099] As used herein, restoring or inducing thymus dependency of T cell precursors that mature into mature T cells means either or both of increasing the number of functional mature T cells of recipient origin in the recipient, or providing mature, functional donor T cells to the recipient by providing donor thymic tissue in which T cells can mature. This increase can be partial, an increase that does not result in levels of mature, functional T cells at levels that result in an essentially normal immune response, or partial, e.g., an increase that fails to result in levels of mature, functional T cells in the recipient at levels that result in an essentially normal immune response.

[0100] In certain embodiments, preparation of a recipient for either organ transplantation or thymus replacement comprises any or all of the following steps, which may be performed in the following order:

[0101] First, a preparation of equine anti-human thymocyte globulin (ATG) is intravenously injected into the recipient. The antibody preparation eliminates mature T cells and natural killer (NK) cells. If not eliminated, mature T cells may promote the rejection of both the thymus transplant and the xenograft organ after sensitization. The ATG preparation also eliminates natural killer (NK) cells. NK cells likely have no effect on the transplanted organ but may immediately act to reject the newly introduced thymic tissue. While previous preparations of porcine ATG have had lower titers than equine ATG, anti-human ATG derived from any mammalian host, such as porcine-produced ATG, can also be used. ATG is superior to anti-NK monoclonal antibodies, as the latter generally do not lyse all host NK cells, whereas polyclonal ATG mixtures can lyse all host NK cells. However, anti-NK monoclonal antibodies can be used. In individuals with relatively severe immunocompromise, this step is not necessary. As host (or donor) T cells mature in the xenogeneic thymus, they will become tolerant to the thymic tissue. Alternatively, as the host immune system gradually recovers, it may be desirable to treat the host to induce tolerance to the thymic tissue.

[0102] Optimally, the recipient's thymus can be removed. In a thymectomized recipient, recipient T cells do not have the opportunity to differentiate in the recipient's thymus, but must differentiate in the hybrid thymus tissue. In some cases, the recipient may need to be splenectomized to avoid anemia.

[0103] Second, the recipient can be given low doses of radiation. While this step is thought to be beneficial in bone marrow transplants (by creating hematopoietic space for newly infused bone marrow cells), it is less important in thymus transplants without bone marrow transplants. However, sublethal doses, e.g., doses approximately equal to or greater than 100 but less than about 400 rads, total body radiation, and 700 rads of local thymus radiation can be used.

[0104] Third, natural antibodies can be adsorbed from the recipient's blood. Antibody removal can be achieved by exposing the recipient's blood to antigens of the donor or donor species, for example, by hemoperfusion of the donor's liver to adsorb the recipient's natural antibodies. Preformed natural antibodies (nAbs) are the primary agents of graft rejection. Natural antibodies bind to xenogeneic endothelial cells and are primarily of the IgM class. These antibodies are independent of any known prior exposure to xenogeneic donor antigens. The B cells that generate these natural antibodies tend to be T cell-independent and are usually tolerized to self-antigens by exposure to these antigens during development. Again, this step may not be necessary, at least initially, in patients with relatively severe immunocompromise.

[0105] The hybrid thymus tissue is transplanted into the recipient, which may include fetal or neonatal liver or spleen tissue.

[0106] Any combination of one or all of these procedures may support the survival of transplanted thymus tissue or another xenogeneic organ.

[0107] The methods of the present disclosure can be used to confer tolerance to a xenograft, for example, where the transplant donor is a non-human animal, e.g., a pig, e.g., a minipig, and the transplant recipient is a primate, e.g., a human.

[0108] The xenograft donor and the individual providing the tolerance-inducing thymic tissue may be the same individual or may be as closely related as possible, for example, it is preferable to derive xenografts from highly or completely inbred donor colonies.

[0109] The second mammalian species (i.e., the donor) can be a non-human mammalian species, such as a porcine species (e.g., a miniature pig species) or a non-human primate species. Non-limiting examples of the first mammalian species include pigs, rodents, non-human primates, cows, goats, and horses.

[0110] In one embodiment, the second mammalian species (i.e., the donor) is an at least partially inbred miniature pig (e.g., the pig is homozygous at the swine leukocyte antigen (SLA) locus and / or homozygous at at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of all other loci). Genetic engineering can be performed on fully or partially inbred pigs (e.g., minipigs, transgenic pigs, etc.). For example, inbred Massachusetts General Hospital (MGH) minipigs may be used in the methods of the invention. These include MGH minipigs that have been inbred for 40 years and are homozygous at all loci. In one embodiment, inbred SLA dd Miniature swine may be used. Mezrich et al. and Sachs, Histocompatible miniature swine: An inbred large-animal model, Transplantation, 2003;75:904-907. Pigs from the National Swine Resources and Research Center (NSRRC, RADIL, University of Missouri, Columbia MO) may also be used in the methods of the invention.

[0111] The first mammalian species (i.e., recipient) can be a primate, such as a non-human primate (e.g., a baboon or a cynomolgus monkey), or a human. In one embodiment, the second species is human.

[0112] In various embodiments, the donor (second species) and recipient (first species) are of different species, for example, the donor is a non-human animal, such as a minipig, and the recipient is a human.

[0113] Methods of transplanting a graft from such a donor animal of a second mammalian species into a recipient mammal of a first mammalian species (eg, a human) are also encompassed by the present disclosure.

[0114] The cells, tissues, organs, or bodily fluids of the transgenic donor animals can be used for transplantation (e.g., xenotransplantation). Grafts harvested from the donor animals for transplantation can include, but are not limited to, heart, kidney, liver, pancreas, lung transplant, intestine, skin, thyroid, bone marrow, small intestine, trachea, cornea, limbs, bone, endocrine glands, blood vessels, connective tissue, progenitor stem cells, blood cells, hematopoietic cells, pancreatic islets, brain cells, and cells, bodily fluids from endocrine and other organs, and combinations thereof.

[0115] The cell can be any type of cell, hi some embodiments, the cell is a hematopoietic cell (e.g., a hematopoietic stem cell, a lymphocyte, a bone marrow cell), a pancreatic cell (e.g., a beta islet cell), a kidney cell, a cardiac cell, or a liver cell.

[0116] Bone marrow cells (BMC) from the donor animal, or hematopoietic stem cells (eg, fetal liver suspension or mobilized peripheral blood stem cells) may be infused into the recipient.

[0117] The method may also include one or more of the following treatments: treatments that inhibit T cells, block complement, or otherwise downregulate the recipient's immune response to the graft.

[0118] Treatments to promote graft tolerance and / or reduce immune recognition include the use of immunosuppressants (e.g., cyclosporine, FK506), antibodies (e.g., anti-T cell antibodies such as polyclonal antithymocyte antiserum (ATG) and / or monoclonal anti-human T cell antibodies such as LoCD2b), irradiation, and methods to induce mixed chimerism. (See U.S. Patent Nos. 6,911,220, 6,306,651, 6,412,492, 6,514,513, 6,558,663, and 6,296,846.) Kuwaki et al., Nature Med., 11(1):29-31, 2005. Yamada et al., Nature Med., 11(1):32-34, 2005.

[0119] In some embodiments, the recipient is thymectomized and / or splenectomized. Thymic irradiation can be used.

[0120] In some embodiments, the recipient is administered a low dose of radiation (e.g., a sublethal dose of 100 rads to 400 rads of total body irradiation). Local thymic radiation may also be used.

[0121] The recipient can be treated with a drug that depletes complement, such as cobra venom factor.

[0122] Natural antibodies can be eliminated by organ perfusion and / or transplantation of tolerance-inducing bone marrow. Natural antibodies can be absorbed from the recipient's blood by hemoperfusion of the donor species' liver. Cells, tissues, or organs used for transplantation can be genetically modified so that they are not recognized by the host's natural antibodies (e.g., cells are α-1,3-galactosyltransferase deficient).

[0123] In some embodiments, the methods include treatment with a human anti-human CD154 mAb, mycophenolate mofetil, and / or methylprednisolone. The methods can also include agents useful for supportive care, such as anti-inflammatory agents (e.g., prostacyclin, dopamine, ganciclovir, levofloxacin, cimetidine, heparin, antithrombin, erythropoietin, and aspirin).

[0124] In some embodiments, donor stromal tissue is administered.

[0125] Immunosuppressants, also called immunosuppressive drugs, can be any compound that reduces the function or activity of one or more aspects of the immune system, such as the humoral or cellular immune system or components of the complement system.

[0126] Non-limiting examples of immunosuppressants include: (1) antimetabolites such as purine synthesis inhibitors (inosine monophosphate dehydrogenase (IMPDH) inhibitors, e.g., azathioprine, mycophenolic acid, and mycophenolate mofetil), pyrimidine synthesis inhibitors (e.g., leflunomide and teriflunomide), and antifolates (e.g., methotrexate); (2) calcineurin inhibitors such as tacrolimus, cyclosporin A, pimecrolimus, and voclosporin. (3) TNF-α inhibitors such as thalidomide and lenalidomide; (4) IL-1 receptor antagonists such as anakinra; (5) mammalian target of rapamycin (mTOR) inhibitors such as rapamycin (sirolimus), deforolimus, everolimus, temsirolimus, zotarolimus, and biolimus A9; (6) corticosteroids such as prednisone; and (7) antibodies against any one of several cellular or serum targets (including antilymphocyte globulin and antithymocyte globulin).

[0127] Non-limiting exemplary cellular targets and their respective inhibitor compounds include complement component 5 (e.g., eculizumab); tumor necrosis factor (TNF) (e.g., infliximab, adalimumab, certolizumab pegol, afelimomab, and golimumab); IL-5 (e.g., mepolizumab); IgE (e.g., omalizumab); BAYX (e.g., nerelimomab); interferons (e.g., faralimomab); IL-6 (e.g., ercilimomab); IL-12 and IL-13 (e.g., lebrikizumab and ustekinumab); CD3 (e.g., muromonab-CD3, otelixizumab, teplizumab, visilizumab); CD4 (e.g., clenoliximab, keliximab, and zanolimumab); CD11a (e.g., efalizumab); CD18 (e.g., erulizumab); CD 20 (e.g., afutuzumab, ocrelizumab, pascolizumab); CD23 (e.g., lumiliximab); CD40 (e.g., teneliximab, toralizumab); CD62L / L-selectin (e.g., acelizumab); CD80 (e.g., galiximab); CD147 / basigin (e.g., gavilimomab); CD154 (e.g., ruplizumab); BLyS (e.g., belimumab); These include, but are not limited to, CTLA-4 (e.g., ipilimumab, tremelimumab); CAT (e.g., bertilimumab, lerdelimumab, metelimumab); integrins (e.g., natalizumab); IL-6 receptor (e.g., tocilizumab); LFA-1 (e.g., odulimomab); and IL-2 receptor / CD25 (e.g., basiliximab, daclizumab, inolimomab).

[0128] The recipient's natural antibodies can be eliminated by organ perfusion and / or tolerance-inducing bone marrow transplantation.

[0129] In one embodiment, the recipient is treated with a preparation of equine anti-human thymocyte globulin (ATG) injected intravenously (e.g., about 25-100 mg / kg, e.g., 50 mg / kg, e.g., 3, 2, or 1 day before transplantation). The antibody preparation eliminates mature T cells and natural killer cells. The ATG preparation also eliminates natural killer (NK) cells. Anti-human ATG obtained from any mammalian host can also be used. Additionally, if further T cell depletion is indicated, the recipient can be treated with a monoclonal anti-human T cell antibody, such as LoCD2b (Immerge BioTherapeutics, Inc., Cambridge, Mass.). For bone marrow transplantation, the recipient can be administered low-dose radiation. Optionally, the recipient can be treated with a complement-depleting agent, such as cobra toxin factor (Cobra Toxin Factor), e.g., 1 day before transplantation.

[0130] In some embodiments, maintenance therapy (e.g., initiated immediately prior to transplant and continued for at least several days after transplant) includes treatment with a human anti-human CD154 mAb. Mycophenolate mofetil (MMF) can be administered to maintain whole blood levels. Methylprednisolone may be administered beginning the day of transplant and then tapered over the next 3-4 weeks.

[0131] Various agents useful for supportive care (eg, days 0-14) include anti-inflammatory agents such as prostacyclin, dopamine, ganciclovir, levofloxacin, cimetidine, heparin, antithrombin, erythropoietin, and aspirin.

[0132] In some embodiments, donor stromal tissue is administered. It may be obtained from fetal liver, thymus, and / or fetal spleen and transplanted into the recipient, for example, within a renal capsule. Thymus tissue can be prepared for transplantation by embedding it under the autologous renal capsule for revascularization. Stem cell engraftment and hematopoiesis across the interspecies barrier can be enhanced by providing a hematopoietic stromal environment from the donor species. The stromal matrix provides species-specific factors, such as hematopoietic growth factors, adhesion molecules, and their ligands, necessary for interactions between hematopoietic cells and their stromal environment.

[0133] Because the liver is the primary site of hematopoiesis in the fetus, fetal liver can also serve as an alternative to bone marrow as a source of hematopoietic stem cells. Each organ contains an organ-specific stromal matrix that can support the differentiation of its respective undifferentiated stem cells transplanted into the host. As an alternative or adjunct to implantation, fetal liver cells can be administered in a fluid suspension.

[0134] Bone marrow cells (BMCs), or another source of donor hematopoietic stem cells, such as fetal liver suspensions, can be infused into the recipient. The donor BMCs have the appropriate site in the recipient and grow and proliferate continuously with the remaining host cells, forming a chimeric lymphocyte hematopoietic population. This process exposes newly forming B cells (and the antibodies they produce) to donor antigens, so that the graft will be recognized as self. Tolerance to the donor is also observed at the T cell level in animals in which hematopoietic stem cell, e.g., BMC, engraftment has been achieved. The use of xenogeneic donors allows the feasibility of using bone marrow cells and organs from the same animal or from genetically matched animals.

[0135] Example The present invention will be better understood from the following Experimental Details, however, those skilled in the art will readily appreciate that the specific methods and results described are merely illustrative of the invention, as more fully described in the claims that follow.

[0136] Example 1 - Creation of a human / pig hybrid thymus to achieve immune tolerance to porcine antigens with optimal immune function Strong immune responses to xenografts are difficult to control with conventional immunosuppression without excessive toxicity. Thymus transplantation is a promising approach to induce T cell tolerance for xenotransplantation. Humanized mice generated using human hematopoietic stem cells (HSCs) and swine (SW) thymus grafts have previously been shown to be tolerant to both species. However, this approach still faces several challenges. First, T cells selected on SW MHC in the swine thymus may not optimally recognize antigens presented by surrounding human MHC (HLA). Second, SW thymic epithelial cells (TECs) do not present human tissue-specific self-antigens (TRAs), which may impair negative selection and may lack Tregs specific for human TRAs. These issues were overcome by generating the human / swine hybrid thymus described herein.

[0137] method To generate human / pig hybrid thymocytes, thymic stromal cells were isolated by digestion of human fetal (20-week gestational age) and pediatric (4-month-old) thymuses with Liberase, followed by magnetic depletion of human CD45+ cells. Human CD45+ cells were resuspended in Matrigel, treated with 2-deoxyglucose to inhibit glycolysis, and injected into frozen / thawed fetal SW thymus tissue, resulting in a reduced number of pig thymocytes in the fetal SW thymus (Figure 2A-C).

[0138] These injected SW thymi were then transplanted into irradiated NOD scid common γ-chain knockout (NSG) mice, followed by injection of human fetal liver-derived CD34 HSCs from the same huTEC donor or an allogeneic donor. At 12–20 weeks posttransplant, the transplanted thymi were excised, sectioned, and stained to detect human TECs using two-photon confocal microscopy (Figure 1A–1D). Because the number of TECs in the adult thymus decreases due to thymic atrophy, huTECs and thymic mesenchymal cells (TMCs) derived from the thymus of a 17-year-old donor were grown on a 2D Matrigel matrix, and the cells were injected into fetal pig thymus tissue and subsequently transplanted into humanized mice (Figure 1E–1H).

[0139] result huTEC-injected SW thymi were functional in humanized mice and supported human thymus formation. Cytokeratin (CK) 14+HLA-DR+ cells and CK8+HLA-DR+ cells, as well as CK8+CK14+HLA-DR+ cells, were detected in hybrid thymi generated from both human fetal and pediatric donors. These TECs were widely distributed and intermingled with porcine TECs (Figures 1A-1D). EpCAM+ TECs from 17-year-old thymuses were expanded fivefold in a single passage. Hybrid thymi generated using in vitro-expanded human TECs and mesenchymal cells contained human TECs (Figures 1E-1H).

[0140] conclusion Injection of human thymic stromal cells into the porcine thymus is an effective technique for generating human / pig hybrid thymi. Human TECs from old thymi could be expanded in vitro using the protocol described herein and were detectable long-term in porcine grafts.

[0141] Example 2 - Development of a method for generating hybrid thymus Injection method The cells were resuspended in Matrigel to prevent leakage from the pig thymus after injection. In principle, human PBMCs were used instead of human thymic epithelial cells in this experiment. First, 10 million human PBMCs were stained with CFSE (2.5 μM) as a tracking dye. CFSE-stained PBMCs (8 million cells) were resuspended in 140 μl of Matrigel on ice to a cell concentration of 50,000 cells per μl. Three different methods were used to inject cells into thawed pig fetal thymus fragments: Method A: Place the pieces into the wells of a V-bottom 96-well plate (5–8 μl) and inject using a Hamilton syringe; Method B: Injection using PE50 tube (20 μl) Method C: Injection (4-6 μl) using a Hamilton syringe while holding the piece outside the well with forceps until the Matrigel solidifies.

[0142] All injected pieces were transferred to different wells of a 96-well plate containing DMEM / F12 medium supplemented with 10% FBS. After 3 hours, the pieces were digested with Liberase. The number of released cells was determined by flow cytometry, and CFSE-stained injected PBMCs were tracked. As shown in Figure 3A, at least a portion of the injected cells was recovered for all three injection methods.

[0143] In summary, injected cells were retained in pig thymus tissue when resuspended in Matrigel before injection. Matrigel is the trade name for a gel-like protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and produced and sold by Corning Life Sciences and BD Biosciences.

[0144] A method for depleting porcine thymocytes to prevent rejection of human thymic stromal cells There are different strategies for ex vivo depletion of thymocytes in porcine thymic fragments, including anti-porcine CD3 immunotoxin and complement-mediated toxicity (complement plus rabbit serum as a source of anti-porcine CD2). However, these two strategies were ineffective. The former strategy induced apoptosis in porcine thymocytes at higher concentrations, killing SP and DP T cells as well as non-T cells, leaving a large population of viable thymocytes in the thymus. The latter strategy was not effective for ex vivo depletion of porcine thymocytes in thymic fragments. Results not shown.

[0145] Considering these results, the following reagents were tested to deplete porcine thymocytes ex vivo using the following method: Day 0: Thaw porcine thymus pieces and incubate with one of the following (in triplicate): a) Cyclosporin A b) Hydrocortisone c) Notch inhibitors (γ-secretase inhibitors = GSi) d) ABT-737 e) 2-deoxyguanosine f) 2D glucose g) Untreated At different time points, tissues were digested and examined for the percentage of dead and apoptotic cells (see Figure 3B).

[0146] Ex vivo conditioning with 2-deoxyglucose (2DG, 100 mM) for 12 hours was the best method for depleting thymocytes while preserving stromal cells. The ratio of remaining viable double-positive CD4 and CD8 cells (DP) or single-positive (SP) CD4 (SP-CD4) or SP-CD8 cells to double-negative (DN) cells, including stromal cells, was used as the readout. Treatment with 100 nM 2DG for 12 hours resulted in the lowest ratio and was therefore the best strategy for depleting thymocytes while preserving stromal cells. See Figure 3C.

[0147] Preparation of hybrid thymus Using 2-DG treatment and a Hamilton syringe to deplete pig thymocytes for infusion of human thymic stromal cells (Method C, above), hybrid thymi were generated and tested in vivo. Specifically, two vials of fetal pig thymus were thawed. After pipetting up and down to release as many thymocytes as possible, half of the pieces were treated with 100 mM 2-DG for 12 hours, while the other half was left untreated.

[0148] The next day, six vials of human fetal thymus were thawed. After pipetting up and down to release as many thymocytes as possible, Liberase was used to digest and release stromal cells to create a single-cell suspension. After depletion of human CD45+ cells using magnetic-activated cell sorting (MACS), 10 million thymic stromal cells were dissolved in 150 μl of cold Matrigel at a concentration of 66,000 cells per μl. Approximately 10 μl of cells (approximately 660,000 thymic stromal cells) were injected into each fetal pig thymus piece (either 2DG-treated or untreated). The pieces were held with fine forceps at room temperature for approximately 2 minutes until the Matrigel solidified. Each piece was then transferred to a well of a 96-well plate containing medium containing 10% human serum. As a control, some pieces were not injected with human thymic stromal cells. After 10 minutes in the incubator, the plates were transferred, still on ice, to a mouse facility for transplantation into immunodeficient NSG mice. Because the recipient mice had previously been thymectomized, they lacked a native mouse thymus, and the only site for thymus formation was the transplanted thymus fragment. First, recipient NSG mice were irradiated (100 cG) and subsequently injected with human fetal liver-derived CD34+ hematopoietic stem cells (HSCs). Then, one thymus fragment was transplanted under the renal capsule of each recipient mouse.

[0149] The experimental design is shown in Figure 4.

[0150] Every 2-3 weeks after transplantation, blood was collected from the mice to assess the level of human immune cell reconstitution. As shown in Figure 5, treatment of the porcine thymus fragments with 2-DG did not affect human thymus formation in the transplanted thymus.

[0151] At 20 weeks after transplantation, mice were euthanized, and the transplanted thymus was removed and frozen in optically coagulated tissue (OCT). After cryosectioning, slides were stained with antibodies against human HLA-DR, cytokeratin 8 (CK8, as a marker for cortical thymic epithelial cells (TECs)), and CK14 (as a marker for medullary TECs). Because the CK antibody is cross-reactive with both human and porcine TECs, HLA-DR was used to differentiate human and porcine TECs. As shown in Figure 6, the injected human TECs mixed with porcine TECs were detected within the transplanted thymus. HLA-DR+ cells that were negative for CK were HSC-derived antigen-presenting cells that migrated from the bone marrow to the transplanted thymus.

[0152] The results of in vitro T cell expansion suggested that peripheral T cells from mice with hybrid thymuses were partially tolerant to the human TEC donor (Figure 7B).

[0153] Human T cells from all mice were tolerant to porcine donor dendritic cells (DCs), but not to the porcine DCs from the third group of SLA-CC (Fig. 7A).

[0154] Human T cells from all mice were tolerant to human HSC-donor DCs (Fig. 7B).

[0155] Human T cells from mice with a pig thymus proliferated at similar levels in response to both human thymic donor-DCs (allogeneic with human HSCs) and human allogeneic-DCs. Human T cells from mice with a hybrid thymus proliferated at reduced levels in response to human thymic donor-DCs compared with allogeneic-DCs (Figure 7B).

[0156] Example 3 - Increased responsiveness to human tissue-specific autoantigens (TRAs) (MART-1, NYESO1, and pancreatic islet antigen IA-2) in human T cells developing in the porcine thymus (SW / HU mice) compared to cells developing in the human thymus (HU / HU mice) method To evaluate the hypothesis of the lack of negative selection of human TRA-specific T cells developing in the pig thymus, we generated two groups of humanized mice using the same human fetal liver CD34+ HSCs and either autologous human fetal thymus (HU / HU) or pig fetal thymus (SW / HU). See Figure 8A. In both groups, the native mouse thymus was removed to ensure that thymopoiesis occurred exclusively in either the human or pig thymus. Approximately 22 weeks after transplantation, mice were euthanized, and pooled lymph node (LN) and splenocytes were depleted for mouse CD45+ cells using MACS. The remaining cells were cocultured with autologous HSC-derived dendritic cells along with different human TRA proteins to measure T cell proliferation in response to these TRAs.

[0157] result As shown in Figure 8B, human peripheral T cells in SW / HU mice demonstrated significantly increased proliferative responses to human TRAs (IA-2, MART-1, and NYESO1) presented by autologous human DCs. The amino acid sequences of these TRAs differ significantly between humans and pigs. This finding supports the lack of negative selection of human TRA-specific T cells in the pig thymus and demonstrates the need for the use of a hybrid thymus.

[0158] Example 4 - Lower survival of human Treg and CD8 T cells developed in the porcine thymus (SW / HU mice) compared to cells developed in the human thymus (HU / HU mice) method The mice (SW / HU and HU / HU mice) prepared in Example 3 were euthanized approximately 24 weeks after transplantation. The transplanted thymus and pooled spleens and lymph nodes (cervical, axillary, brachial, and mesenteric LNs) were collected. Thymocytes, splenocytes, and LN cells were isolated by physical force (crushing the thymus tissue between two slides and crushing the spleen and LNs through a 70 μm cell strainer using a syringe plunger). RBCs in splenocytes were lysed using ACK lysis buffer (Gibco). Isolated cells were counted using a hemocytometer. After counting the total cell number, 0.2 to 1 million cells from each thymus and pooled spleens and LNs were stained with the antibodies shown in Figure 9 for flow cytometry analysis. Cells were read using a BD Fortessa flow cytometry instrument, and data were analyzed using Flowjo software.

[0159] result The numbers of spleen and LN cells and transplanted thymocytes were significantly higher in HU / HU mice compared with SW / HU mice (Figures 9A and 9G). The ratio of DP (double-positive CD4+CD8+) to SP cells (single-positive SP-CD4 or SP-CD8) in the transplanted thymus was similar between HU / HU and SW / HU mice (Figure 9B). A functional thymus should have a higher ratio of DP than SP cells.

[0160] Furthermore, the fraction of Tregs (regulatory T cells) in SP-CD4 cells was similar (Figure 9C). The levels of proliferating (Ki67+) SP-CD4 and Treg cells were higher in SW / HU thymus compared with HU / HU thymus (Figures 9D and 9F). Furthermore, a higher fraction of SW / HU thymic Tregs expressed CD45RO (Figure 9E).

[0161] In contrast to similar levels of SP-CD4 and SP-CD8 cells in the thymus, SW / HU mice had lower CD8 cells among T cells and lower proportions of Tregs among CD4 cells in the periphery (spleen and lymph nodes, Figures 9I–9K). This finding indicated that both of these cell subsets require interaction with the same MHC that selected for their survival. Higher rates of naive memory conversion were evident in both peripheral CD4 and CD8 cell subsets in SW / HU mice (Figures 9L and 9M). Ki67+ (proliferation) (Figure 9O), HLA-DR+ (activation) (Figure 9N), CD45RO+ (Figure 9P), and CTLA-4+ cells (Figure 9Q) within peripheral CD4, CD8, and Treg cells did not differ between HU / HU and SW / HU mice.

[0162] These results further demonstrate the need for the use of hybrid thymus.

[0163] Example 5 - Generation of hybrid thymus with embryonic stem cell-derived TECs (ES-TECs) and long-term persistence of human TECs in the hybrid thymus method 2×10 5 Human fetal or pediatric thymic stromal cells (hu-TECs) or hPSC-TEC precursors (1–2 × 10) were intravenously injected with human fetal liver-derived CD34+ cells before transplantation under the renal capsule of thymectomized immunodeficient NSG (Nod / Scid / Ilr2g- / -) mice. 5 Human TECs were injected into fetal pig thymus tissue (SW thymus). As a control, fetal pig thymus tissue without human TECs was transplanted.

[0164] Approximately 20 weeks after transplantation, the transplanted thymus was removed, sectioned, and stained to detect human TECs using two-photon confocal microscopy.

[0165] The cells were then released from the stroma and the number of cells was determined by flow cytometry.

[0166] result As shown in Figure 10, human TECs in the "hybrid thymus" were long-term (>20 weeks) persistent. As shown in Figure 10A, all grafts (upper left panel), including pig thymuses that were not injected with human TECs, contain human HSC-derived HLA-DR APCs (green) and pig / human CK14 TECs (red). However, only the hybrid thymus contained detectable HLA-DR CK14 human TECs (yellow, see arrow).

[0167] Flow cytometry staining of gated CD45-negative cells in digested stroma from long-term thymic grafts showed the presence of EPCAM+, CD105-negative hu-TECs only in human thymus (Fig. 10B, top right) and SW grafts injected with hPSC-TEC precursors (Fig. 10B, bottom left panel), but not in uninjected SW THY grafts (Fig. 10B, top left). See Fig. 10C.

[0168] Example 6 - Injection of hES-TECs into the porcine thymus promotes T cell development and peripheral CD4 + and CD8 + Increased T cells method 2×10 5 hES-TECs (1–2 × 10) were injected intravenously with human fetal liver-derived CD34 cells before transplantation under the renal capsule of thymectomized immunodeficient NSG (Nod / Scid / Ilr2g) mice. 5 Human TECs were injected into fetal pig thymus tissue (SW thymus). As a control, fetal pig thymus tissue was transplanted without human TECs.

[0169] Splenocytes and thymocytes from thymic grafts were analyzed by flow cytometry at 18-22 weeks post-transplant.

[0170] result As shown in Figures 11A-D, hES-TEC-injected thymic grafts expressed higher absolute numbers of human splenic CD3+ cells in splenic mononuclear cells. + T cells, CD8 + T cells, CD4 + T cells and CD45RA+ CCR7 + Recent thymic emigrated CD31 cells defined + CD4 + with untreated cells.

[0171] To assess the final stage of terminal differentiation, thymocytes were stained for expression of HuCD45, CD19, CD14, CD4, CD8, CD45RA, and CD45RO. As shown in Figure 11E, hES-TEC-injected thymic grafts contained higher numbers of total thymocytes, as well as higher numbers of human CD45 cells, double-positive CD4+CD8+, single-positive CD4+CD8-, CD4-CD8+, and immature CD45RO+.

[0172] The scope of the present invention is not limited to what has been specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the example materials, configurations, structures, and dimensions shown. Numerous references, including patents and various publications, have been cited and described in the description of this invention. The citation and description of such references is provided merely to clarify the description of the invention and is not an admission that the references are prior art to the invention described herein. All references cited and described herein are incorporated herein by reference in their entirety. Variations, modifications, and other embodiments of what is described herein will occur to those skilled in the art without departing from the spirit and scope of the invention. While certain embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the invention. The foregoing is offered by way of example only, and not by way of limitation.

Claims

1. 1. An isolated hybrid thymic tissue comprising thymic epithelial cells from a first mammalian species and thymic tissue from a second mammalian species, wherein (a) the first mammalian species is a primate or a human, (b) the thymic epithelial cells are produced from induced pluripotent stem cells (iPSCs) or embryonic stem cells, and (c) the second mammalian species is a pig.

2. 2. The isolated hybrid thymus tissue of claim 1, wherein the first mammalian species is human.

3. The isolated hybrid thymus tissue of claim 1 or 2, wherein the pig is a miniature pig.

4. The isolated hybrid thymus tissue of any one of claims 1 to 3, wherein the thymus tissue from the second mammalian species is fetal thymus tissue or neonatal thymus tissue.

5. The isolated hybrid thymic tissue of any one of claims 1 to 4, wherein the thymic epithelial cells are produced from iPSCs.

6. The isolated hybrid thymic tissue of any one of claims 1 to 4, wherein the thymic epithelial cells are produced from embryonic stem cells.

7. 5. The isolated hybrid thymic tissue of any one of claims 1 to 4, wherein the thymic epithelial cells are produced from embryonic stem cells that share HLA alleles with the first mammalian species.

8. 5. The isolated hybrid thymus tissue of any one of claims 1 to 4, wherein the embryonic stem cells have been genetically engineered to share HLA alleles with the first mammalian species.

9. 9. The isolated hybrid thymic tissue of any one of claims 1 to 8, wherein the hybrid thymic tissue is produced by introducing thymic epithelial cells from the first mammalian species into the thymic tissue from the second mammalian species.

10. 10. The isolated hybrid thymic tissue of any one of claims 1 to 9, wherein the hybrid thymic tissue is produced by injecting thymic epithelial cells from the first mammalian species into thymic tissue from the second mammalian species.

11. The hybrid thymus tissue is isolated by the following steps: (i) treating thymus tissue from the second mammalian species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from the first mammalian species into the 2DG-treated thymic tissue. The isolated hybrid thymus tissue according to any one of claims 1 to 10, which is produced by a method comprising:

12. 12. The isolated hybrid thymic tissue of claim 11, wherein in step (ii), the thymic epithelial cells are suspended in Matrigel before being injected into the 2DG-treated thymic tissue.

13. 1. A composition comprising thymic epithelial cells from a first mammalian species, thymic tissue from a second mammalian species, and biological material for obtaining isolated hybrid thymic tissue, wherein (a) the first mammalian species is a primate or a human, (b) the thymic epithelial cells are produced from induced pluripotent stem cells (iPSCs) or embryonic stem cells, and (c) the second mammalian species is a pig.

14. 14. The composition of claim 13, wherein the first mammalian species is a human.

15. The composition of claim 13 or 14, wherein the thymic epithelial cells are produced from iPSCs.

16. The composition of claim 13 or 14, wherein the thymic epithelial cells are produced from embryonic stem cells.

17. The composition according to any one of claims 13 to 16, wherein the thymic epithelial cells are suspended in the biomaterial, which is preferably Matrigel.

18. 17. The composition of any one of claims 13 to 16, wherein the biomaterial is selected from polyethyleneimine and dextran sulfate, poly(vinylsiloxane) copolymer polyethyleneimine, phosphorylcholine, poly(ethylene glycol), poly(lactic-co-glycolic acid), poly(lactic acid), polyhydroxyvaleric acid and copolymers, polyhydroxybutyrate and copolymers, polydioxanone, polyanhydrides, poly(amino acids), poly(orthoesters), polyesters, collagen, gelatin, cellulose polymers, chitosan, alginate, fibronectin, extracellular matrix proteins, vinculin, agar, agarose, hyaluronic acid, matrigel, and combinations thereof.

19. The composition according to any one of claims 13 to 18, further comprising hematopoietic stem cells.

20. 1. An in vitro or ex vivo method for generating hybrid thymic tissue, comprising: (i) treating thymus tissue from a second mammalian species with 2-deoxyglucose (2DG); and (ii) introducing thymic epithelial cells from a first mammalian species into the 2DG-treated thymic tissue.

1. A method comprising: (a) the first mammalian species is a primate or a human; (b) the thymic epithelial cells are generated from induced pluripotent stem cells (iPSCs) or embryonic stem cells; and (c) the second mammalian species is a pig.

21. 21. The method of claim 20, wherein in step (ii), the thymic epithelial cells are suspended in Matrigel before being injected into the 2DG-treated thymic tissue.

22. 22. The method of claim 20 or 21, wherein the first mammalian species is human.

23. The method according to any one of claims 20 to 22, wherein the pig is a miniature pig.

24. The method according to any one of claims 20 to 23, wherein the thymus tissue is fetal thymus tissue or neonatal thymus tissue.

25. The method of any one of claims 20 to 24, wherein the thymic epithelial cells are generated from iPSCs.

26. The method of any one of claims 20 to 24, wherein the thymic epithelial cells are prepared from embryonic stem cells.

27. 27. The method of claim 26, wherein the thymic epithelial cells are generated from embryonic stem cells that share HLA alleles with the first mammalian species.

28. 28. The method of claim 27, wherein the embryonic stem cells are genetically engineered to share HLA alleles with the first mammalian species.

29. 29. The method of any one of claims 20 to 28, wherein the hybrid thymic tissue is produced by introducing thymic epithelial cells from the first mammalian species into thymic tissue from the second mammalian species.

30. 30. The method of any one of claims 20 to 29, wherein the hybrid thymic tissue is produced by injecting thymic epithelial cells from the first mammalian species into thymic tissue from the second mammalian species.

31. A pharmaceutical composition comprising the hybrid thymus tissue of any one of claims 1 to 12 for inducing tolerance in a recipient mammal of a first species to a graft obtained from a donor mammal of a second species.

32. A pharmaceutical composition comprising the hybrid thymus tissue of any one of claims 1 to 12 for restoring or inducing immunocompetence in a recipient mammal of a first species.

33. 13. A pharmaceutical composition comprising a hybrid thymic tissue according to any one of claims 1 to 12 for restoring or promoting thymus dependency of T cell precursor cells that develop into mature functional T cells in a recipient mammal of a first species.

Images